The purpose of this project is to determine whether DNA sequence differences in the promoter regions of the several human globin genes are relevant to their developmental regulation. Our studies have focused on the gamma globin gene promoter. Truncation deletion mutants ranging from 1200 to 55 nucleotides upstream from the start site of transcription have been created by recombinant DNA methods. Linker scanning mutants in which a synthetic linker replaces portions of the promoter have been constructed so as to delete and/or replace conserved sequences within the promoter region. Both deletion and linker scan mutant genes have been introduced into HeLa cells to quantitate their expression. Deletion of increasing amounts of the gamma gene promoter results in progressive decrease in promoter function. In contrast, deletion of one of the two conserved "CCAAT" regions in the promoter results in a 2-6 fold increase in promoter function. The normal gamma and beta globin genes and the various deletion and linker scan mutants of the gamma gene promoter region have been linked to a gene that confers neomycin resistance. These have been introduced into a hematopoietic cell line. Baseline and induced gamma globin gene expression will be measured to determine whether specific DNA regions in the promoter are crucial for its developmental regulation.